Molecular Vision 2003; 9:60-73 Received 31 December 2002 | Accepted 26 February 2003 | Published 11 March 2003
© 2003 Molecular Vision
Three-dimensional analysis of mouse rod and cone mitochondrial cristae architecture: Bioenergetic and functional implications Guy A. Perkins,1,2 Mark H. Ellisman,1,2 Donald A. Fox3,4,5 1
Department of Neurosciences and 2National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA; 3College of Optometry, 4Department of Biology and Biochemistry, and 5Department of Pharmacology and Pharmaceutical Sciences, University of Houston, Houston, TX Purpose: Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. Methods: We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen different measurements of mitochondrial substructures were analyzed. Photoreceptor oxygen consumption was determined in dark- and light-adapted retinas. Results: Rod and cone mitochondria displayed an orthodox conformation. Cone inner segments, compared to rods, contained 2-fold more mitochondria and were more CO reactive. Rod and cone mitochondria had similar outer-inner membrane width, contact site width, diameter and density, crista width, number of cristae/volume, number of cristae segments/ volume, and fraction of cristae with multiple segments. In contrast, cone mitochondria had narrower crista junctions, greater cristae connectivity, and approximately 3-fold more cristae membrane surface area compared to rods. The increased cristae membrane surface area in cones was accomplished by connecting more cristae segments together, rather than by creating more cristae. Conclusions: These results demonstrate that middle wavelength (M) cones have a different bioenergetic signature than do rods and suggest that the aerobic ATP demand and production is greater in light-adapted cones than in light-adapted rods. Cones utilize two complimentary strategies to increase their aerobic ATP production: increase the number of mitochondria and increase the cristae surface membrane area. The greater ATP generation by cones may also provide increased protection against metabolic insults and apoptosis compared to rods.
The basic structure, biochemistry and function of mammalian retinal rod and cone photoreceptors are well described [1-7]. Biochemical, physiological, and microscopy studies have established that 60-65% of retinal mitochondria are located in the photoreceptor inner segments, that the inner segments have the highest retinal cytochrome c oxidase (CO) activity (i.e., stain more intensely), and that photoreceptors have 2 to 3 fold greater oxygen consumption than the inner retina [8-14]. Although rod and cone photoreceptors share several common features and properties, many of their functions are significantly different. Compared to rods, cones are less sensitive to light, respond and recover more quickly, adapt over an extended range of luminance intensities, have higher relative permeability for Ca2+ in their outer segments (COSs), have a higher fraction of the dark current carried by Ca2+, and express higher levels of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) [9,15-21]. Moreover, in pri-
mate retinas the cone inner segments (CISs), except for the narrow CISs in the foveal region [14,22], have significantly more mitochondria, have higher CO activity, and stain more intensely for Na+,K+-ATPase than rod photoreceptor inner segments (RISs) [8,9,14]. These results suggest that cones are bioenergetically more active than rods and thus should produce more ATP than rods. There are relatively few reports on the ultrastructural details of rod and cone mitochondria and even fewer on their comparative aspects [8,14,23-25]. One of the earliest ultrastructural studies of retinal mitochondria focused on guinea pig RISs and found that the major component of RISs was elongated and densely packed mitochondria [26]. In primates, CISs contain approximately 10 times more mitochondria than RISs and the mitochondrial volume density is approximately 60% in rod ellipsoids and approximately 80% in cone ellipsoids [8,14]. Prior to the finding that photoreceptors responded to light with graded potentials [6], Sjostrand [23] suggested that the relative number of mitochondria in rods and cones reflected a difference in the cells’ mean frequency of discharge. Subsequently, Cohen [8] suggested that the cones had more
Correspondence to: Donald A. Fox, Ph.D., University of Houston, College of Optometry, 505 J. Davis Armistead Building, Houston, TX, 77204-2020; Phone: (713) 743-1964; FAX: (713) 743-2053; email:
[email protected] 60
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mitochondria because their energy requirements were significantly greater than those of rods, which is consistent with the above noted conclusion. More recently, it was suggested that the numerous cone mitochondria enhance the waveguide prop-
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erties of cones [14]. To date, however, the three-dimensional (3-D) architecture of photoreceptor inner segment mitochondria and the relationship between the density and arrangement of cristae and mitochondrial bioenergetics has not been adequately studied. The 3-D technique with the highest resolution available for studying mitochondria is electron microscope tomography [27-29]. Conventional transmission electron microscopy (TEM) played an early and essential role in formulating and shaping the concepts of mitochondrial bioenergetics [30-32]. More recently, the development and refinement of 3-D electron imaging using high-voltage electron tomography has provided significant new insights into mitochondrial bioenergetics as well as the ultrastructure and substructure of normal and apoptotic mitochondria [33-40]. The limitation of conventional TEM operating at 100 kV is that it only provides single images from ultrathin (50-100 nm thick) sections. This does not permit adequate sampling from mitochondria with dimensions that are typically 0.2 to 2 µm or resolution of mitochondrial substructures that range from 2-50 nm. In con-
Figure 1. Electron microscopy and photoreceptor oxygen consumption of 21 day old mouse retina. Conventional electron micrographs of retinas and photoreceptor oxygen consumption (QOPR) from postnatal day 21 mice. A: Electron micrographs of the rod (r) and cone (c) outer and inner segments and distal outer nuclear layer from superior temporal retina of a light-adapted mouse. Note that the tips of the middle wavelength-sensitive cone outer segments lie in the rod inner segment region. The cone inner segment diameter is approximately twice that of the rod inner segment. The cone nuclei are located in the distal portion of the outer nuclear layer and contain several clumps of irregularly shaped heterochromatin, whereas the rod nuclei contain a single compact mass of heterochromatin. Scale bar represents 10 µm. B: Transverse section through ellipsoid region of photoreceptor inner segment illustrating cytochrome oxidase stained rod (r) and cone (c) mitochondria. Note that there are approximately twice as many cone mitochondria per photoreceptor compared to rod photoreceptors and that the cone mitochondrial inner membranes stain more intensely for cytochrome oxidase than do the rods. Scale bar represents 1 µm. C: QOPR. QOPR was measured in pairs of dark-adapted and rod saturating light-adapted whole retinas from five mice. 61
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trast, electron microscopy (EM) tomography utilizes thick sections (0.25-5.0 µm), microscopes operating at 400-3000 kV, multiple tilt-series usually ranging from -60° to +60°, and sophisticated computer algorithms for precise spatial localization [33,35,36,38]. Our goals were to determine, in light-adapted mouse retinas, the: (1) number of mitochondria and oxidative enzyme activity in RIS and CIS, (2) photoreceptor oxygen consumption (QOPR) and compare it to that in dark-adapted photoreceptors, (3) dimensions and connectivity of RIS and CIS mitochondrial cristae and contact sites, and (4) structural motifs of RIS and CIS cristae. Here we show that mouse CIS, compared to RIS, mitochondria have: approximately 2-fold more mitochondria, higher oxidative enzyme staining, narrower crista junctions, greater cristae connectivity and approximately 3-fold more cristae membrane surface area. The implications for mitochondrial ATP generation and response to metabolic dysfunction are discussed. METHODS Materials: All chemicals were analytical or molecular biology grade and were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Experimental animals and retinal fixation: All experimental and animal care procedures were in compliance with the principles of the American Physiological Society, the NIH Guide for the Care and Use of Laboratory Animals and Maintenance (NIH publication No. 85-123, 1985) and were approved by the Institutional Animal Care Committee of the University of Houston. C57BL/6 mice (Harlan Sprague Dawley, Indianapolis, IN) were housed in a room maintained at 22 ± 1 °C with a 12:12 h light dark cycle and cage illumination of 5-10 lux. Mice were mated and upon giving birth, postnatal day 0, the litters were culled to eight pups. Only female mice were used in order to directly compare our present results with previous results [39,41-44]. On postnatal day 21 (weaning), two hours after light onset, the mice were sacrificed by decapitation. The eyes were Figure 2. Quantitative analysis of mitochondrial substructures. A: Use of XVoxtrace. The software program XVoxtrace allows volume segmentation, as shown here for a cone mitochondrion. Segmentation is based on membrane topography and separates individual cristae, inner boundary and outer membranes. The separate tracings of three cristae are shown in yellow, red and blue. B: Use of Synuview. Using the software program Synuview, individual or any combination of cristae surfaced-rendered can be displayed in order to visualize their 3-D shapes and membrane architecture in user-defined orientations. This program was used to classify the structural motifs of lamellae, tubes, crista junctions, and constrictions. A crista showing all of these elements is displayed in yellow. The outer membrane is shown in blue. C: Use of XDend. Measurements of surface-rendered elements permitted a comparison of substructures inside mitochondria using the program XDend. A contoured crista is shown on the left. On the right, a table of numerical values associated with volume, surface area, length, and number of contours is displayed for the crista on the left. 62
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removed and fixed by immersion in ice-cold 3% glutaraldehyde, 2% paraformaldehyde and 0.1% CaCl2 in 0.1 M cacodylate buffer (pH 7.4) without or with (only for CO histochemistry) 4% sucrose. As described [41], the superior portion of the eye was identified with a suture, the corneas were cut at the limbus and each eye was placed in 15 ml of the same fresh fixative for either 2 h (for CO histochemistry) or overnight at 4 °C for conventional EM and EM tomography. For all experiments, the superior temporal retina, 200250 µm from the optic nerve head, was used because it contains almost exclusively middle wavelength-sensitive (M) cones [45]. We wanted to compare these middle wavelengthsensitive cones, which are similar to those in other mammals [45,46], to rods. The number and distribution pattern of middle wavelength-sensitive cones is adult-like by postnatal day 14 and is similar in male and female mice [45]. Conventional EM and CO histochemistry: For conventional EM, each fixed eye was placed in fresh ice-cold buffer and the superior temporal quadrant near the optic nerve (central retina) was trimmed. The retinal sections were dehydrated and embedded in Spurrs resin as described [44]. Ultra-thin longitudinal sections of the whole retina and transverse sections through the RIS and CIS were stained with uranyl ac-
© 2003 Molecular Vision
etate and lead before being examined in a JEOL 100-C or 1200EX transmission electron microscope (Tokyo, Japan). The number of mitochondria per cross-section of RIS and CIS was obtained from 10-30 photoreceptors from each of five different mice. The mean number from each mouse was determined and then an overall mean ± SEM was calculated (n=5). For CO histochemistry, the cornea, lens and vitreous were removed and the eyecups were cryoprotected in 30% sucrose at 4 °C for an additional 24 h. The retinas were quick frozen, 15-25 µm sections were cut on a microtome, incubated, histochemically reacted for CO, and the superior temporal retinal sections processed for EM examination as described [47,48]. The differences in staining intensity between rod and cone mitochondria were evaluated qualitatively on transverse sections through the RIS and CIS of four different mice. The specificity of the cytochemical reaction was determined by adding 10 mM KCN to the incubation buffer, which completely eliminated any CO reaction product in the mitochondria (data not shown). Photoreceptor oxygen consumption: QOPR was recorded from retinas isolated from postnatal day 25 mouse retinas. Both retinas were placed in the recording chamber at the same time. QOPR was determined polarographically and recorded continu-
Figure 3. Perpendicular extensions connect tubular cristae. This figure demonstrates that tubular cristae in cone mitochondria connect to the inner boundary membrane through perpendicular extensions from the tubular shaft. A: Serial slices through the tomographic reconstruction of a cone mitochondrion. The serial slices illustrate how the crista membrane extends from the tubular shaft towards the periphery of the mitochondrion, forms a crista junction (arrow) and finally contracts away from the boundary membrane. Scale bar represents 50 nm. B: Volume segmentation and surface rendering of the same crista. The two 90° rotation views (top and bottom) highlight the dimensions of the crista junction opening (arrows) in relation to the crista (yellow) and mitochondrial inner membrane (blue). Crista junctions (*) from other cristae also are shown and are indicated by fenestrations in the inner membrane. These fenestrations accurately portray the size and variation in dimensions of these junctions. 63
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per crista. Overall, measurements from tomographic reconstructions were made from 28 distinct mitochondria (14 from rods and 14 from cones) using retinas from four different mice. EM tomography: double-tilt series: Double-tilt tomography improves the accurate reconstruction of the 3-D organization of complex biological structures [50]. The most recognized advantage of double-tilt reconstruction is the improved resolution in the Z-dimension (perpendicular to the specimen plane). A second, less-appreciated advantage is that, unlike single-tilt tomography, double-tilt tomography provides optimal resolution for elongated structures, such as tubular cristae, at any orientation in the specimen. A third advantage is the capability to correct distortions that arise during the acquisition of images. For example, if the specimen shrinks nonuniformly during image collection, the resultant distortions can be corrected more effectively during registration of two related reconstructions than during alignment of the respective tilt series. Double-tilt tomography was performed by first collecting two tilt series around orthogonal axes. After the first tilt series was complete, the specimen grid was rotated 90° and
ously in the dark or during presentation of a rod-saturating light adapting stimulus as described [12,49]. The mean dry weight of each retina was 0.55 mg. EM tomography: Single-tilt series: For EM tomography, the superior temporal quadrant retinal sections were dehydrated, embedded in Durcupan resin and imaged in situ using the techniques described by Perkins and co-workers [33,34]. 3-D reconstructions were obtained from semi-thick samples (approximately 500 nm) in a tilt-series every 2° from -60° to +60° on a JOEL 4000EX electron microscope operated at 400 kV. Fourteen different types of measurements of mitochondrial substructures were obtained with large sampling sizes: (1) outer membrane to inner (OM-IM) widths, (2) cristae widths, (3) cristae junction diameters, (4) contact site widths, (5) contact site diameters, (6) classical contact site density, (7) bridge contact site density, (8) number of cristae segments per mitochondrial volume, (9) number of cristae per mitochondrial volume, (10) cristae volume per mitochondrial volume, (11) fraction of cristae with multiple segments, (12) cristae surface area per mitochondrial surface area, (13) number of cristae segments per crista, and (14) number of constrictions Figure 4. Cone mitochondria have high cristae connectivity. A: Slice through a cone mitochondrion with one traced crista. Connectivity is defined as the sum of continuous connections of cristae segments with each other that form a complete crista. In other words, the connection of well-defined cristae shapes, such as tubes and lamellae, is determined in 3-D. Electron tomography provided this 3-D mapping analysis, which is not feasible with conventional electron microscopy of thin sections. In one 2.2 nm slice of the volume (shown), the yellow tracings highlight all the branches of the largest crista that has the most segments and constrictions of all the cone mitochondrial reconstructions. The connectivity was best mapped after the volume was segmented and surface-rendered. Scale bar represents 50 nm. B and C: Perpendicular views after segmentation of the crista. The crista, traced in A, is shown in yellow. It appears almost maze-like because of its great connectivity of more than 20 segments. The outer membrane is shown in blue. This largest crista measured in cone mitochondria extends through roughly half of the cone mitochondrial volume, has a volume of 110,000 nm3, which represents approximately 9% of the entire mitochondrial volume, and has an inner membrane surface area of 490,000 nm2, which is equivalent to 97% of its total outer membrane surface area. This same connectivity and extent was not observed in rod mitochondria (Figure 5). D: “Loop” connectivity. An unusual crista architecture in cone mitochondria was observed that was rarely noted in rod mitochondria. This architecture is described as loop connectivity because of the connection of crista segments following a closed pathway through the intracristal space back to each other rather than to the inner boundary membrane. Four loops from different cone mitochondria are illustrated. The arrows indicate the narrow connections (constrictions) between loop segments. 64
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refining 3-D transformation. Densities were linearly scaled to match between volumes. The combination of volumes was conducted in Fourier space. The 3-D Fourier transform of each volume was computed and for every point containing data from both volumes, the values were averaged. Otherwise, the values from one or the other volume were used. The inverse of this composite Fourier transform provided the final combined volume. Statistical analysis: All group data were analyzed by twotailed Student’s t-test. All data are presented as means ± SEM. For all data, the difference between rods and cones was regarded as significant if p
